One of the most important goals in oncology is the identification and elimination of treatment resistant cells; hypoxic cells are the most familiar examples of this type of cell. Kennedy, el al., Biochem. Pharm. 1980, 29, 1; Moulder, et al., Int. J. Radioat. Oncol. Biol. Phys. 1984, 10, 695; Adams, Cancer, 1981, 48, 696. Hypoxic cells are seldom found in normal tissues, and are generally found only in conjunction with certain tumors, vascular diseases, or after a stroke.
As certain tumors enlarge, the tissue often outgrows its oxygen and nutrient supply because of an inadequate network of functioning blood vessels and capillaries. Although the cells deprived of oxygen and nutrients may ultimately die, at any given time a tumor may produce viable hypoxic cells. These hypoxic cells, although alive, have very low oxygen concentrations because of their remoteness from the blood vessels.
The level of molecular oxygen has important implications in disease diagnosis and prognosis. In medical oncology, for example, hypoxic cells in solid tumors may be highly resistant to killing by some forms of chemotherapy. When chemotherapeutic agents are administered to patients, the agents are carried through the functioning blood vessels and capillaries to the target tissue. Because hypoxic tissue lacks a fully functioning blood supply network, the chemotherapeutic drugs may never reach the hypoxic cells; instead, intervening cells scavenge the drug. The result is that the hypoxic cells survive and recurrence of the tumor is possible. Kennedy, et al., supra.
Tissue hypoxia also hinders the effectiveness of radiation therapy, especially of neoplasms. Radiation treatment is most effective in destroying oxygen containing cells because oxygen is an excellent radiation sensitizer. The presence of hypoxic cells impedes this treatment because their low oxygen concentration renders the ionizing radiation relatively ineffective in killing the cancerous cells. Therefore, hypoxic cells are more likely to survive radiation therapy and eventually lead to the reappearance of the tumor. The importance of hypoxic cells in limiting radiation responsiveness in animal tumors is well known, Adams, supra; Moulder, et al., supra; Chapman, et al., "The Fraction of Hypoxic Clonogenic Cells in Tumor Populations," in Biological Bases and Clinical Implications of Tumor Radioresistance 61, G. H. Fletcher, C. Nevil, & H. R. Withers, eds., 1983. Studies have revealed that such resistant cells greatly affect the ability of radiation and chemotherapy to successfully sterilize tumors in animals. Substantial work since that time has shown similar problems in human tumors. Despite the progress in animal studies regarding the identification of hypoxic cells, limited success has been achieved in humans. One reason for this disparity may relate to differences in tumor growth and other host related factors, but in addition, there has been no suitably accurate method to assess tissue oxygen at a sufficiently fine resolution.
Venous oxygen pressure is generally .about.35 Torr, an oxygen level providing nearly full radiation sensitivity. As the oxygen level decreases below 35 Torr, radiation resistance gradually increases, with half-maximal resistance at about 3.5 Torr, and full resistance at about 0.35 Torr. Therefore, it is necessary to measure much lower oxygen levels than are usually encountered in normal tissue. Current technology does not meet this need. Oxygen partial pressure measured using current techniques often yields an average value for large numbers of neighboring cells. This is a severe impediment for detection and diagnosis because histological evaluation of solid tumors suggest that important changes in cellular oxygen can occur over dimensions of even a few cell diameters. Urtasun, el al., Br. J. Cancer, 1986, 54, 453. Nitroheterocyclic drugs have been under extensive investigation as oxygen indicators. It is known that this class of compounds has the potential for resolution at the cellular level and can provide sufficient sensitivity to monitor the low oxygen partial pressures described above. This technique involves the administration of nitroaromatic drugs to the tissue of interest. The drugs undergo bioreductive metabolism at a rate which increases substantially as the tissue's oxygen partial pressure decreases. The result of this bioreductive metabolism is that reactive drug products are formed which combine chemically to form adducts with predominantly cellular proteins. Because the metabolic binding of these compounds to cellular macromolecules is inhibited by oxygen, these compounds bind to hypoxic cells in preference to normal, healthy, oxygen-rich tissue. This preferential metabolic binding, or adduct formation, provides a measure of the degree of hypoxia. Koch, et al., Int. J. Radiation Oncology Biol. Phys., 1984,10, 1327.
Misonidazole (MISO) 3-methoxy-1-(2-nitroimidazol-1-yl)-2-propanol, and certain of its derivatives have been under extensive investigation as indicators of hypoxia in mammalian tissue. Chapman, et al., Int. J. Radiat. Oncol. Biol. Phys., 1989,16, 911; Taylor, et al., Cancer Res., 1978, 38, 2745; Varghese, et al., Cancer Res., 1980, 40, 2165. The ability of certain misonidazole derivatives to form adducts with cellular macromolecules, referred to as binding throughout this application, has formed the basis of various detection methods.
For example, .sup.3 H or .sup.14 C labeled misonidazole has been used in vitro and in vivo, with binding analyzed by liquid scintillation counting or autoradiography. Chapman, 1984 supra; Urtasun, 1986, supra; Franko, et al., Cancer Res., 1987, 47, 5367. A monofluorinated derivative of misonidazole has utilized the positron emitting isotope F18 for imaging bound drug in vivo, Rasey, et al., Radiat. Res., 1987, 111,292. The method of the preparation of the PET derivative of ethanidazole was described in Tewson T. J. Synthesis of [.sup.18 F] Fluoroetanidazole: a potential new tracer for imaging hypoxia. Nuclear Medicine & Biology, 24(8):755-60, 1997.
A hexafluorinated derivative of misonidazole (1-(2-hydroxy-3-hexafluoro-isopropoxy-propyl)-2-nitroimidazole has been assayed directly (no radioactive isotopes) via nuclear magnetic resonance spectroscopy (NMR or MRI) techniques. Raleigh, et al., Int. J. Radiat. Oncol. Biol. Phys., 1984, 10, 1337. Polyclonal antibodies to this same derivative have allowed immunohistochemical identification of drug adducts. Raleigh, et al., Br. J. Cancer, 1987, 56, 395. An iodine isotope has been incorporated into another azomycin derivative, azomycin arabinoside, allowing radiology techniques of detection. Parliament, et al., Br. J. Cancer, 1992, 65, 90.
A fluorescence immunohistochemical assay for detecting hypoxia is described in the literature. Raleigh, et al., 1987, supra. A method for preparing immunogenic conjugates for use in such assays is broadly disclosed in U.S. Pat. No. 5,086,068, issued to Raleigh, et al., on Feb. 4, 1992 ("Raleigh patent"). The Raleigh patent describes a method for preparing an immunogenic conjugate comprising a known fluorinated misonidazole derivative and an immunogenic carrier protein, hemocyanin. The compound used in this method (CCI-103F) was a hexafluorinated derivative of 2-nitroimidazole misonidazole, described above in connection with NMR studies.
The resulting conjugate is used to raise rabbit polyclonal antibodies specific for the misonidazole derivative. Fluorescence immunohistochemical studies showed that the polyclonal antibodies bound to hypoxic (central) regions of spheroids (a multicellular aggregate of cells in tissue culture having some properties more closely related to tumors) and tumor sections in patterns similar to those revealed by audioradiographic studies using radioactive drug alone, i.e. without polyclonal antibodies.
However, polyclonal antibodies are plagued by numerous difficulties such as cross-reactivity, lack of specificity, insensitivity, inability to purify the actual antibodies of interest, and highly unstable supply.
The Raleigh patent's technology, of conjugating a small antigen to a large carrier protein to elicit an immune response, is a central basis of antibody production and is well known in the art. Those skilled in the art would also appreciate that nitroaromatics must be activated by chemical or biochemical reduction to cause adducts to form with cellular macromolecules. Further, it has not been possible to produce monoclonal antibodies using the methods described in the Raleigh patent and paper (Raleigh et al.,1987, supra).
The Raleigh patent discloses immunogenic conjugates useful for producing polyclonal antibodies, but data generated using the patent's teachings has produced variable results, problematic in a detection technique. Furthermore, independent experimentation performed according to the Raleigh patent's methods did not reproduce the high degree of conjugation between the misonidazole derivatives and the protein as was claimed. See, e.g., U.S. Pat. No. 5,540,908, the disclosures of which are herein incorporated by reference in their entirety.
The bioreductive drug assays described above do not directly measure oxygen partial pressure, even though this is the required value, using the example of radiation therapy to predict radiation response. Rather, the assays measure adduct formation, a biochemical process which is inhibited by oxygen. The data generated using these methods has shown that the degree of inhibition by oxygen varies substantially from tissue to tissue. Franko, et al., 1987, supra. Furthermore, the maximum rate of adduct formation in the complete absence of oxygen is also highly variable from tissue to tissue, as is the maximum percentage of inhibition by oxygen, Koch, in Selective Activation of Drugs by Redox Processes, Plenum Press, pp. 237-247, Adams, et al., eds, New York, 1990. Another way of expressing these limitations is that the bioreductive formation of nitroaromatics provide only a relative indication of varying oxygen levels, but is inadequate at providing an absolute measurement of oxygen partial pressure because there are several factors which affect adduct formation in addition to changes in oxygen, non-oxygen-dependent factors. Additionally, the choice of nitroaromatic drug affects the variability related to the non-oxygen-dependent factors.
Early research efforts (i.e., before the invention claimed in U.S. Pat. No. 5,540,908 on Nov. 19, 1992) had focused on misonidazole and certain of its derivatives. However, misonidazole is the most susceptible of several drugs tested to non-oxygen-dependent variations in adduct formation. Koch, Selective Activation, supra. Other problems relate to various physicochemical properties of existing drugs, all of which can influence the non-oxygen dependent variations in adduct formation. For example, the hexafluorinated misonidazole derivative described above had a high degree of insolubility.
Although 2-nitroimidazoles labeled with radiochemical tracers such as tritium and .sup.14 C provide a sensitive method for detecting tissue hypoxia using autoradiographic methods, the biohazards and costs associated with these techniques are a significant drawback. The amount of radioactivity associated with the administration of such labeled drugs, which still requires a tissue biopsy, becomes a substantial problem in animal studies and an even greater problem in humans where 30 millicuries of tritiated drug are typically used. Urtasun, et al., 1986, supra. .sup.14 C is prohibitively expensive and causes unacceptable radiation exposures. The use of such radioactive tracers is generally not acceptable because of the stringent requirements associated with handling radioactive tissues and bodily fluids. There are also practical limitations to the use of radioactive tracers. For example, the delay required for audioradiographic analysis of the tissue sections, often several weeks, is a very serious impediment to the rapid analysis required in treatment determination. Moreover, toxicity problems associated with certain misonidazole derivatives resulted in the drug being administered at a relatively low concentration, which decreased detection sensitivity. Thus, to utilize the high sensitivity of radioactive drug methods, short-lived isotopes analyzable by non-invasive methods such as PET and SPECT are preferred; there is still a need for such methods.
Many human and animal diseases are characterized by the pathological formation of tissue hypoxia and ischemia. Hypoxic cells in solid tumors have been associated with treatment resistance by radiation, Moulder, supra, and some forms of chemotherapy, Kennedy, supra. Treatment of such conditions can only be optimized by determining the extent and degree of hypoxia in the affected tissues of individual patients. Accordingly, there is a great oncological need to identify hypoxic cells.
While biopsy-based methods are applicable to many forms of analysis in tumors, non-invasive assays are required for diseases of normal tissue such as heart attack and stroke. Again, one must employ techniques such as MRS/MRI, PET, and SPECT.
Previous studies have exemplified the determination of hypoxia in normal and diseased tissues by detecting metabolites of drugs named 2 (2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide (hereinafter referred to as EF5) and 2(2-nitro-1H-imidazol-1-yl)-N-(3,3,3-trifluoropropyl) acetamide (hereinafter referred to as EF3). See U.S. Pat. No. 5,540,908, issued to Koch et al, the disclosures of which are herein incorporated by reference.
Notwithstanding the significant advances already attained with EF5 and EF3, there still remains a need in the art for compounds that are useful in noninvasive imaging techniques, such as MRI and PET. See also Detection of Hypoxic Cells by Monoclonal Antibody Recognizing 2-Nitroimidazole Adducts, Cancer Res., 1993, 53, 5721-76, the disclosures of which are herein incorporated by reference. It is highly desirable to be able to assay for the presence of hypoxic cells in an animal or human tumor, and to do so predictably and without the concomitant hazards associated with radioactivity. The compounds and methods of the claimed invention address these, as well as other, needs in the art.